Dissecting the impact of dietary fiber type on atherosclerosis in mice colonized with different gut microbial communities

Dietary fiber consumption has been linked with improved cardiometabolic health, however, human studies have reported large interindividual variations in the observed benefits. We tested whether the effects of dietary fiber on atherosclerosis are influenced by the gut microbiome. We colonized germ-free ApoE−/− mice with fecal samples from three human donors (DonA, DonB, and DonC) and fed them diets supplemented with either a mix of 5 fermentable fibers (FF) or non-fermentable cellulose control (CC) diet. We found that DonA-colonized mice had reduced atherosclerosis burden with FF feeding compared to their CC-fed counterparts, whereas the type of fiber did not affect atherosclerosis in mice colonized with microbiota from the other donors. Microbial shifts associated with FF feeding in DonA mice were characterized by higher relative abundances of butyrate-producing taxa, higher butyrate levels, and enrichment of genes involved in synthesis of B vitamins. Our results suggest that atheroprotection in response to FF is not universal and is influenced by the gut microbiome.


Introduction
Individual responses to the same diet or therapeutic drugs are often inconsistent and not observed that atheroprotection was associated with butyrate production and enrichment for bacterial genes involved in pathways for carbohydrate metabolism and vitamin synthesis. detected in one diet-bound group but not the other ( Supplementary Fig. 2c). Eleven weeks after dietary 121 treatment, cecal samples were collected and used to assess terminal microbial communities. By the end 122 of the experiment, 5 of the genera were no longer detected in cecal contents of mice on either diet, while 123 4 genera (Clostridium, Faecalibacterium, Gemmiger, and an undetermined Ruminococcus genus) were 124 found only in FF-fed mice ( Supplementary Fig. 2c). This introduces the possibility that the differences 125 observed in the assembled communities between dietary groups for DonA mice, are the result of 126 inconsistent engraftment rather than the effect of diet. Alternatively, since microbial communities 127 undergo considerable fluctuations in the period after colonization 31 , it is possible that these missing taxa 128 were present in the CC-bound mice, but below detectable levels. This scenario is supported by the fact 129 that i) all of the missing taxa were detected in the human donor sample used to inoculate all DonA mice, 130 and ii) similar diet-driven patterns were observed with Faecalibacterium and Gemmiger abundances in a diversity (Shannon) was higher in mice colonized with DonA consuming FF relative to CC but was not was significantly increased with the FF diet in DonA mice but was reduced by FF feeding in DonB mice (Fig.   147 1g).

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Mice in all treatment groups were dominated by Firmicutes and Bacteroidetes and had detectable 149 levels of Proteobacteria, and Verrucomicorbia (Fig. 1d). Actinobacteria were detected in FF-fed DonA 150 mice, CC-fed DonB mice, and both diets within DonC mice. FF feeding lowered the Bacteroides to

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Firmicutes ratio for all donors (Fig. 1e), but the only significant reduction was observed in DonA. There 152 was generally little consistency in the diet-associated enrichment patterns observed across all donor 153 groups, even when considering phylum level changes, suggesting a lack of a universal response to dietary 154 treatment (Fig. 1c,d).

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We used the Microbiome Multivariable Association with Linear Models (MaAsLin 2) 32 to analyze 156 enrichment patterns at the genus level caused by diet for each donor group. A total of 29 genus-level Although Akkermansia has been reported to thrive on diets poor in fermentable fiber 35 , and our data 169 shows that microbiota composition can influence Akkermansia response to specific fiber sources. It is also  were no observed differences between diets in either DonB-or DonC-colonized mice (Fig. 2a-c). To further 179 characterize atherosclerosis disease status, lesions were assessed for macrophage infiltration by 180 immunohistology with MOMA-2 antibodies. There were no statistically significant differences observed 181 between diets in lesion MOMA-2 density in any of the donor groups (Fig. 2a,d), but there was a trend of 182 reduction with FF feeding observed in DonA mice (P=0.11). This suggests that macrophage infiltration is 183 reduced with FF feeding in DonA-colonized mice. Previous studies observed conflicting results in terms of

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To test whether fermentable fiber consumption altered lipid composition in circulation, we 194 measured plasma levels of total cholesterol, HDL cholesterol and triglycerides. No statistical differences 195 were observed between diets for any of the donor groups for total cholesterol, HDL cholesterol, or 196 triglycerides ( Fig. 2e-g). We also assessed aortic expression levels of Abca1 and Abcg1 mRNA by RT-qPCR 197 as markers of reverse cholesterol transport but did not observe any statistically significant differences 198 between diets within any of the donor groups ( Supplementary Fig. 3d,e). These results suggest that the 199 atheroprotective effect of FF consumption observed in DonA mice was not mediated through major

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There were no significant differences observed in expression of these markers between diets in any of the 204 donor groups ( Supplementary Fig. 3a-c). These data suggest that the atheroprotective effect of FF-feeding 205 in DonA mice may be independent of these immune processes and reverse cholesterol transport, although 206 further analyses are needed to fully rule out this hypothesis. elevated butyrate levels in DonA mice, but reduced butyrate concentrations in DonB-colonized mice 216 compared to CC-fed counterparts (Fig. 3c). Butyrate levels were unchanged between diets in DonC-217 colonized mice. The branched-chain fatty acids isobutyrate and isovalerate, which are primarily produced 218 via protein fermentation, were not significantly affected by diet within any of the donor groups (Fig. 3e,f).

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Total SCFA concentrations (the sum of acetate, propionate, and butyrate) within donor groups were 220 increased by FF-feeding in DonA and DonB, but not DonC (Fig. 3d). These results are consistent with the 221 shifts observed in the microbiota. Among the genera that were increased by FF feeding only in DonA mice 222 were Clostridium, Oscillpspira, Ruminococcus, Gemmiger, and Faecalibacterium, (Fig. 1c) all of which 223 contain butyrate producing species 39,40 . Interestingly, most of these genera were also present in the other 224 donor groups but were not impacted by FF feeding. This could be due to complex, community-level 225 interactions (e.g., competition) influencing responses of individual genera to dietary fiber, or strain-level 226 differences in response to diet, or both. Our data is also consistent with previously reported studies 227 suggesting that butyrate protection against atherosclerosis in mice occurs in the absence of major changes

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Although we observed increased cecal propionate levels in the athero-protected mice in DonA, we did not 232 detect differences in plasma cholesterol levels. The concentration and location of propionate within the 233 gastrointestinal tract (i.e., greater concentration in the small intestine when consumed orally vs. greater 234 concentration in the large intestine when produced via fiber fermentation) may influence the effect of

Bacterial functional profiles were differentially modulated by dietary fiber in a donor-specific manner.
We next sought to identify links between the functional potential of the microbiome and 241 atheroprotection by examining changes in microbial metagenomic profiles. We performed shotgun 242 sequencing of DNA isolated from cecal contents (average of 29.4 ± 7.7 million paired-end reads/sample; n=5/diet-donor group). Sequence data was analyzed with HUMAnN3 to generate metagenomic functional 244 profiles that included KEGG orthology (KO) abundances for each mouse. Hierarchical clustering of KO 245 profiles using Bray-Curtis dissimilarity shows that the treatment groups are clearly differentiated from 246 one another, but the effect of diet on clustering patterns varied by donor (Fig. 4a). FF-fed mice colonized 247 with DonA and DonC clustered more by diet than by donor group, suggesting a significant level of FF-248 influenced overlap in KO profiles between these two donor groups, while DonB mice on both diets were 249 distinctly clustered from mice of the other donor groups (Fig. 4a). Differential abundances of individual

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We next aimed to gain further insight into how dietary treatment affected the metabolic 258 pathways of the cecal microbial communities in each donor group. We were specifically interested in 259 teasing out pathways that might help to explain the atheroprotection associated with FF feeding in DonA.

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To achieve this, we used the MicrobiomeAnalyst KEGG pathway tool to conduct pathway enrichment 261 analysis in the KOs that were over-represented by FF feeding (adjusted P-value <0.1) relative to their 262 counterparts consuming the CC diet. Similar to the taxonomy results discussed above, we observed a lack of universality among the metagenomic changes in response to diet. Of the 38 KEGG pathways that were 264 detected as significantly overrepresented by FF-feeding in at least one donor group, only three (Pyruvate 265 metabolism, Amino sugar and nucleotide sugar metabolism, and Biosynthesis of amino acids) were 266 observed across all three donor groups (Fig. 4b). In DonA-colonized mice, 27 pathways were significantly 267 overrepresented in the FF diet relative to CC diet. These included pathways involved in vitamin synthesis 273 Both folate and vitamin B12 are involved in the detoxification of homocysteine, a metabolite of 274 methionine metabolism that has been linked to cardiovascular disease 42,43 . A study involving ApoE -/mice 275 with hyperhomocysteinemia found that supplementation with a mixture of folate, vitamin B12, and 276 vitamin B6 protected against atherosclerosis 44 . Moreover, a recent metagenomic study in humans showed 277 that patients with CVD (n=218) had decreased abundance of genes encoding for components of the folate 278 biosynthesis pathway than healthy patients (n=187) 45 . Interestingly, the authors of that study also found 279 that CVD was associated with lower abundances of propionate and butyrate synthesis genes. To gain a 280 more detailed picture of the metagenomic dynamics of these pathways, we compared the differential 281 abundances of the individual KOs involved in folate biosynthesis (KEGG map00790) and anaerobic 282 cobalamin (vitamin B12) biosynthesis (KEGG M00924). In agreement with our enrichment analysis, most 283 of the differentially abundant KOs in both pathways were upregulated by FF feeding in DonA-colonized 284 mice, but not in the other groups (Fig. 4c). Folate and vitamin B12 were supplied in the FF and CC diets at These results suggest that microbial production of vitamins B12 and folate may act as a potential mediator of the atheroprotection associated with FF diet in mice colonized with this community.

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To uncover associations between atheroprotection and fiber metabolism, we determined the 290 level and type of carbohydrate-active enzymes (CAZymes) families between dietary treatments within 291 each donor group using cecal metagenomic data. We detected a set of CAZyme families that were highly 292 abundant in all donor groups and largely unaffected by diet ( Fig. 5a-b, Supplementary Fig. 4). Differential 293 abundance analysis revealed that the CAZyme families which were most significantly affected by diet were 294 relatively low abundance; about 100-fold lower than the highest abundance CAZymes (Fig. 5c). Given the 295 differences in cecal SCFA levels between treatment groups, this suggests that these highly differential,

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The current study has some limitations that should be addressed. First, we observed an imperfect 325 engraftment efficiency from human donor to mouse recipient. As discussed above, we detected 326 differences in the pre-treatment engraftment patterns of DonA mice. Our evidence above suggests that 327 this difference in detection was likely a consequence of the stochasticity of microbial communities shortly 328 (two weeks) after colonization and not a result of differences in inoculation or contamination.

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Nonetheless, this discrepancy introduces the possibility that the differences observed in atherosclerosis 330 within DonA mice were due to inconsistent engraftment rather than response to diet. Another limitation 331 is that our study only used three human donors. A much larger and more diverse cohort of donors would 332 be needed to fully appreciate the breadth of cardiometabolic responses to these diets, but the limited 333 group used here is sufficient to suggest that the athero-modulatory effect of dietary fiber is microbiota-   profiles. In the current study, we used this information to select three human donor samples: microbiota WLS-sample-1 (referred to here as DonB) is characterized by low butyrate production; and donor WLS- were immediately used to gavage recipient mice. A subset of GF mice was colonized by cohousing with 372 mice that had been gavage-colonized with human feces 4 weeks prior. Cohousing is an effective strategy 373 for colonizing germ-free mice and is similar to gavage in terms of microbiota colonization and phenotype 374 transfer 48,49 . We were unable to detect differences in cecal microbial profiles, nor did we observe 375 significant differences in phenotypes between cohoused mice and their gavage-colonized counterparts 376 ( Supplementary Fig. 2a

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We assembled cleaned (trimmed, host-free) reads into contigs with metaSPAdes (version 3.14.0) with 471 multiple k-mer sizes (metaspades.py -k 21, 33, 55, 77). Contigs shorter than 500bp were discarded from 472 further processing. Open reading frames (ORFs; i.e., microbial metagenes), were predicted from 473 assembled contigs via Prodigal70 (version 2.6.3) using Hidden Markov Model (HMM) with default 474 parameters. All predicted genes shorter than 100bp were discarded from further processing. Nucleotide 475 ORF sequences were converted amino acid sequences and were used as input for run_dbcan (version MaAsLin2 effect size    Forward primer sequences for each target gene are nedoted by "-F" and reverse sequences are denoted by "-R".